Herpes simplex virus (HSV) can infect corneal epithelium and result in primary herpetic keratitis. HSV can then establish a latent infection in the trigeminal ganglia, a site from which it can be reactivated leading to replication of the virus and recrudescence of disease, often of increasing severity. The factors involved in the establishment and maintenance of HSV latency, the reactivation of the virulent infection, and the physical state in which the virus exists during latency have been extremely difficult to study. I propose to establish a cell culture model of latency for HSV infection using a murine embryonal carcinoma cell line which can be induced by treatment with retinoic acid to differentiate into several cell types including neurons and glial cells. To characterize the cell types induced, cell-specific antigens will be detected by immunofluorescence and specific marker enzyme activities will be assayed. Procedures for induction of differentiation will be sought which will enhance the production of neurons, the cell type in which HSV appears to establish latency in vivo. Specific conditions of infection with HSV, including timing of infection during differentiation and multiplicity of infection, will be defined which optimize the yield of latently infected cells. To detect the presence of HSV in latently infected cells the expression of viral specific antigens will be monitored by immunofluorescent methods using anti-HSV antibody or antiserum specific for an immediate early viral protein. Further, viral specific DNA will be detected by in situ hybridization using as a probe HSV genomic DNA labeled with 125I by nick translation. A cell culture system as a model for HSV latency has the advantage of being a source of large quantities of cells which are difficult to obtain by primary explant of nervous tissue from animals. By obtaining increased numbers of infected cells the molecular mechanisms involved in control of neural latency can be studied. Future studies could include investigations to determine the physical state of the virus during latency and also the mechanism of control of viral transcription in these cells. A second advantage of this model is that unlike tumor cell lines of neural origin, the differentiated embryonal carcinoma cells have many characteristics of normal, terminally differentiated nerve cells. Further, the appearance of more than one cell type following differentiation allows the study of how HSV interacts with each cell type and how certain cell-cell interactions might contribute to latency. Understanding the control mechanisms involved in latency should make it possible to modify those controls in such a manner that recurrence can be prevented.